The transplantation of organs to patients having organ diseases or defects was at first limited by technical obstacles to transplantation surgery, and later by the lack of effective immunosuppressive agents. As many of these obstacles were overcome, the major limiting factor quickly became the scarcity of suitable donor organs. While public information campaigns have made headway in convincing people of the importance of designating themselves as potential organ donors, there is still a severe shortage of organs for transplantation. Many patients can only wait as their condition worsens, uncertain of whether a suitable organ will become available before they are too ill to benefit from a transplant.
An obvious limit on the number of vital organs for transplantation is the fact that such organs only become available under unusual circumstances: the death of an otherwise relatively healthy person in a manner that does not damage the vital organs. Therefore, vital organs for human allotransplantation (transplantation between individuals of the same species) will likely always be in short supply. Accordingly, xenotransplantation (transplantation between individuals of different species) provides a desirable additional source of organs for transplantation to humans.
In the development of an optimal xenotransplantation system, several factors must be considered. First, a close phylogenetic relationship between the donor and the recipient is preferable to a more distant relationship. For example, for xenotransplantation of a vital organ to a human, a non-primate generally would be a less desirable donor, in terms of phylogenetic relationship, than a primate.
The order Primates is divided into two suborders: the prosimians and the anthropoids. The anthropoids are further divided into two infraorders: the Platyrrhini, or new world monkeys, and the Catarrhini. The Catarrhini are likewise divided into two superfamilies: the old world monkeys and the hominoids. Hominoids include the great apes and humans. According to this classification scheme, the old world monkeys are more closely related to humans than are the prosimians or the new world monkeys, but not as closely related to humans as are the four genera of apes: Hylobates (gibbons), Pongo (orangutans), Gorilla, and Pan (chimpanzees).
A second very important factor in selecting a preferred xeno-species is its amenability to human handling, captive breeding, experimentation, and the like. Generally speaking, old world monkeys are much more easily maintained than are the apes.
Other important factors for an optimal xenotransplant source species include reproductive rate, body size, cost of maintenance, and anatomic and physiologic similarity to humans. The reproductive rate is a function of the average age at which individuals of the species reach sexual maturity, as well as the gestational duration, multiplicity of births, and number of reproductive years. The size of the source species is important because organs that are too small are not always suitable for transplantation into humans. Since baboons, (genus Papio) are the largest of the old world monkeys, and since their anatomic and physiologic characteristics are very similar to those of humans, they represent a desirable combination of the most important factors.
Baboons do, however, present shortcomings of their own as a genuine alternative to allotransplantation. They do not reach sexual maturity until about age 4 or 5, they typically deliver only one offspring per gestation, and they present a potential risk of transmitting erstwhile baboon pathogens to humans. All of these facts make baboons very unlike the familiar laboratory and model system animals that can be multiplied virtually at will, and that are hosts to pathogens that are relatively well known and easily controlled. For all of these reasons, creating a large colony of baboons to provide a significant supply of donor organs would be a slow and very costly process.
An even greater obstacle to the development of baboons as a useful xenotransplantation source species is that, while the most common human histo-blood group is O, baboons of group O are exceedingly rare. The resulting incompatibility of the organs of virtually all baboons with members of the largest human blood group, as discussed in greater detail below, significantly reduces the utility of present captive baboon colonies, as well as almost all baboons in the wild, as good sources of xenotransplant organs for humans as a group.
Therefore, in addition to the scarcity of suitable donor organs, compatibility considerations further limit the potential pool of donors for a particular patient. This is equally true for both allo- and xeno-organ sources.
Incompatible organs are very likely to be rejected. For example, when organs are transplanted across the ABO histo-blood group barrier, there is a high incidence of antibody-mediated rejection. One antibody-mediated form of organ rejection, known as hyperacute vascular rejection, may be quite rapid. In heart and kidney transplants, hyperacute vascular rejection has been estimated to occur in approximately 66% of ABO-mismatched cases. A second type of antibody-mediated rejection is known as accelerated rejection. In some cases of accelerated rejection, an organ recipient generates anti-donor antibodies which may then aberrantly cross react with the recipient's own cells leading, for example, to complications or death brought on by agglutination of the recipient's blood cells arising from his or her own antibodies. In addition to antibody-mediated rejection in its various manifestations, cellular rejection, associated with the cellular immune response, may also occur, albeit more slowly. Cellular rejection may be a risk even in cases where antibody-mediated rejection has been avoided or overcome. Cooper, D. K. C., Ye, Y., Niekrasz, M., Kehoe, M., Martin, M., Neethling, F. A., Kosanke, S., DeBault, L. E., Worsley, G., Zuhki, N., Oriol, R., & Romano, E. (1993) Transplantation 56:769-777 (hereinafter Cooper et al. (1993)).
Several approaches have been proposed to reduce antibody-mediated rejection, some of which may also diminish the extent of cellular rejection. A splenectomy may be performed, and may accompany pre-transplant plasmapheresis, a process that temporarily removes antibodies from the blood. However, both splenectomy and plasmapheresis may nonspecifically depress all immune responses, instead of exclusively blocking the response to ABO incompatibility alone. Cooper et al. (1993).
A more specific approach to A/B antibody removal involves passage of a patient's plasma through an affinity column that displays the specific glycans recognized by anti-A and/or anti-B antibodies. Only A/B antibodies are bound to the column while non-A/B antibodies remain in the plasma as it passes through the column. Another alternative is to competitively occupy the A/B antibodies without removing them from the plasma, by intravenously infusing small carbohydrates to which the antibodies specifically bind, thus selectively inactivating the antibodies that could otherwise mount an undesirable response to the transplanted organ. Of course, any of the above therapies may also be combined with administration of immunosuppressive drugs. Nevertheless, since A/B antibodies develop and are maintained via continuous sensitization by microbial flora in the gastrointestinal tract, the temporary removal or inactivation of A/B antibodies provides no long-term solution. Cooper et al. (1993).
Incompatibility at the histo-blood ABO locus is therefore a major determinant in limiting the suitability of a xeno- or allo-donor organ for a particular recipient. Commonly known to control a person's blood-group, the products of the ABO locus not only affect antigens on erythrocytes, but also on many other cell surfaces, including the epithelium of several important organs. Therefore, if a donor and recipient are not compatible for traditional blood transfusion because of ABO phenotype differences, they will be likewise incompatible for organ transplantation.
The ABO histo-blood group antigens, the basis of blood group, are found in all anthropoid primates. Socha, W. W., & Ruffie, J. (1983) Blood Groups of Primates: Theory Practice, Evolutionary Meaning Alan R. Liss, New York. These antigens, which are terminal carbohydrate structures, can be found both in soluble form and on the surface of a variety of tissues, depending on the species examined. Additionally, antibodies to the non-expressed antigen(s) are universally present, agglutinating mismatched blood and facilitating complement-mediated attack on tissues following transplant. Genetically, the phenotype is controlled by a single locus that can be occupied by three fundamental alleles encoding enzymes with either A or B activity or no activity (group O).
The A and B enzymes are both glycosyltransferases. The enzymes each transfer a different sugar residue to the same core oligosaccharide. The core oligosaccharide may be variable in length, but terminates with a disaccharide of D-galactose (D-Gal) and N-acetyl-D-glucosamine (GlcNAc), which is usually modified to replace the GlcNAc with a residue of L-fucose (L-Fuc). The enzyme product of the A allele, A transferase, specifically catalyzes the transfer of N-acetyl-D-galactosamine (GalNAc) to the core D-Gal, resulting in a branched terminal trisaccharide having both GalNAc and L-Fuc attached to the core D-Gal. This trisaccharide is the A antigen, and is bound by A antibodies. Likewise, the B allele product, B transferase, specifically transfers D-Gal to the same core oligosaccharide. The resulting trisaccharide, the B antigen, has both L-Fuc and D-Gal attached to the core D-Gal.
The human O allele specifies no active enzyme, and the recessive O phenotype occurs in the absence of any active A or B transferase. The oligosaccharide therefore terminates with a D-Gal/L-Fuc disaccharide. This disaccharide structure is known as the H antigen, and is not bound by either A or B antibodies. In the rare Bombay phenotype, the original GlcNAc of the core disaccharide is never replaced by L-Fuc, and so the core disaccharide retains the D-Gal/GlcNAc terminal structure.
Cells with A transferase activity display the A glycan, while cells with B transferase activity instead display the B glycan. Cells with both activities have both types of glycan antigen modifications, and cells with neither A nor B transferase display the H antigen. The A and B antibodies specifically recognize the A and B glycans, respectively. Martinko, J. M., Vincek, V., Klein, D. & Klein, J. (1993) Immunogenetics 37:274-278; see also Yamamoto, F. (1995) Vox Sang 69:1-7 (hereinafter Yamamoto (1995)); and Clausen, H., Bennett, E. P., & Grunnet, N. (1994) Transfus.-Clin.Biol. 2:78-89.
Since cells from histo-blood group O individuals lack both antigens, they are not subject to attack by A/B antibodies, and therefore can be freely transplanted. Thus, blood group O individuals are considered universal donors of both blood and organs. However while blood group O is the most common group in humans, it is very rare in baboons. Socha, W. W., Moor-Jankowski, J., Ruffie, J. (1984) J. Med Primatol. 13:11-40. This has been an impediment to the development of xenotransplantation protocols involving the use of baboon organs. Bailey, L. L., Nehlsen-Cannarella, S.L., Concepcion, W., & Jolley, W. B. (1985) J. Am. Med. Assoc. 254:3321-3329; Bailey, L. L. & Nehlsen-Cannarella, S.L. (1986) Transplant. Proc. 18(Suppl. 2):88-92.
Previous work on the molecular genetics of the ABO system has established the cDNA sequence and genomic structure of the locus in humans, as well as the amino acid residues conferring the different enzymatic activities. Yamamoto, F. & Hakomori, S. (1990) J. Biol. Chem. 265:19257-19262); Yamamoto (1995); Bennett, E. P., Steffensen, R., Clausen, H., Weghuis, D. O., & van Kessel, A. G. (1995) Biochem. Biophys. Res. Comm. 206:318-325; Yamamoto, F., McNeill, P. D., & Hakomori, S. (1995) Glycobiol. 5:51-58. The coding sequence of the transferase is divided into seven exons. Exon 6 and 7 encode the bulk of the enzyme (13% and 65%, respectively), including its active site. In all examined primate species the residues critical for determining donor substrate specificity (i.e., A versus B activity) are found at amino acid positions 266 and 268, Leu vs. Met and Gly vs. Ala, respectively. Martinko et al. (1993); Kominato, Y., McNeill, P. D., Yamamoto, M., Russel, M., Hakomori, S. & Yamamoto, F. (1992) Biochem. Biophys. Res. Comm. 189:154-164 (amino acid position numbering is according to Yamamoto (1995) and Clausen, et al. (1994)).
In humans, blood group O arises from either of two mutations in an A-like background: a frameshift in exon 6 leading to premature termination, or a Gly&gt;Arg mutation in exon 7 at position 268. Yamamoto, F., Clausen, H., White, T., Marken, J. & Hakomori, S. (1990) Nature 345:229-233 (hereinafter Yamamoto et al. (1990)); Yamamoto, F., McNeill, P. D., Yamamoto, M., Hakomori, S., Bromilow, I. M., Duguid, J. K. M. (1993) Vox Sang 64:175-178; Grunnet, N., Steffensen, R., Bennett, E. P. & Clausen, H. (1994) Vox Sang 67:210-215. However, prior to the work disclosed herein, the nature of the allele(s) conferring an O phenotype in baboons was not known.
Because organs of O phenotype baboons would not elicit an ABO-induced rejection, and because of the other advantageous features of baboons as a xenotransplant source species for man, a group O strain of baboons would be of great medical importance. However, since group O baboons are so rare, the process of locating naturally occurring founders for such a strain would be difficult. Moreover, even if it were possible to locate some few group O baboons of both sexes, it would still take many years of breeding to achieve a group O colony of significant size, because of the relatively slow reproduction and maturation of baboons.
Random mating of non-O individuals, and subsequent postnatal screening of offspring to identify those of O phenotype is equally impractical. Given the rarity of the group O phenotype, it may be assumed that the O allele is also relatively rare, unless A/O or B/O heterozygotes are somehow selected for. The following hypothetical model illustrates the impracticality of random breeding and postnatal selection of O homozygotes:
Assuming no selection and random mating, if the frequency of the O phenotype in a population of baboons is 1%, it would be expected that the frequency of the O allele in that population's gene pool is 10%. Assuming that the A and B alleles are of equal frequency (45% each), random mating would be expected to produce the following genotype frequencies
A/A=0.2025 B/B=0.2025 PA1 A/O=0.09 B/O=0.09 PA1 A/B =0.405 O/O=0.01 PA1 A=0.2925 PA1 B=0.2925 PA1 AB=0.405 PA1 O=0.01 PA1 p(A)=0.423 PA1 q(B)=0.423 PA1 r(O)=0.154
resulting in the following phenotypic frequencies
If the A and B (but not AB) individuals in this population were randomly mated, the frequencies of the alleles in the A and B breeding pool would be
and 100 such matings would on average produce only 2 group O offspring [(0.154).sup.2 *100=2.37].
Using the same Hardy-Weinberg model, if the O allele were assumed to be as frequent as 30%, rather than 10%, the O individuals resulting from random mating and no selection would be 9% of the population, and 100 matings of phenotype A and/or B individuals would be expected to produce 10 group O offspring. It is well documented that the O phenotype is rare in baboons. Given the rarity of the O phenotype, it seems doubtful that the frequency of the O allele is as great as 30%.
Regardless of the actual allele frequency, the above exercise demonstrates that conventional breeding involving matings of non-O individuals can make, at best, incremental progress toward the establishment of a stock of group O baboons. Accordingly, there is a need for a prenatal, or even pre-mating, selection that identifies and favors the O alleles and enhances the rate of production of group O offspring. The invention disclosed herein provides a way to greatly accelerate the establishment of a group O strain of baboon for medical use.